title: Recombinant expression of aryl hydrocarbon receptor for quantitative ligand-binding analysis.
abstract: Recombinant expression of the aryl hydrocarbon receptor (AhR) yields small amounts of ligand-binding-competent AhR. Therefore, Spodoptera frugiperda (Sf9) cells and baculovirus have been evaluated for high-level and functional expression of AhR. Rat and human AhR were expressed as soluble protein in significant amounts. Expression of ligand-binding-competent AhR was sensitive to the protein concentration of Sf9 extract, and coexpression of the chaperone p23 failed to affect the yield of functional ligand-binding AhR. The expression system yielded high levels of functional protein, with the ligand-binding capacity (Bmax) typically 20-fold higher than that obtained with rat liver cytosol. Quantitative estimates of the ligand-binding affinity of human and rat AhR were obtained; the Kd for recombinant rat AhR was indistinguishable from that of native rat AhR, thereby validating the expression system as a faithful model for native AhR. The human AhR bound TCDD with significantly lower affinity than the rat AhR. These findings demonstrate high-level expression of ligand-binding-competent AhR, and sufficient AhR for quantitative analysis of ligand binding.
authors.affiliation: School of Biology, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors.forename: Ming Qi
authors.initials: MQ
authors.lastname: Fan
authors.forename: Alex R
authors.initials: AR
authors.lastname: Bell
authors.forename: David R
authors.initials: DR
authors.lastname: Bell
authors.forename: Sally
authors.initials: S
authors.lastname: Clode
authors.forename: Alwyn
authors.initials: A
authors.lastname: Fernandes
authors.forename: Paul M D
authors.initials: PM
authors.lastname: Foster
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Fry
authors.forename: Tao
authors.initials: T
authors.lastname: Jiang
authors.forename: George
authors.initials: G
authors.lastname: Loizou
authors.forename: Alan
authors.initials: A
authors.lastname: MacNicoll
authors.forename: Brian G
authors.initials: BG
authors.lastname: Miller
authors.forename: Martin
authors.initials: M
authors.lastname: Rose
authors.forename: Osama
authors.initials: O
authors.lastname: Shaikh-Omar
authors.forename: Lang
authors.initials: L
authors.lastname: Tran
authors.forename: Shaun
authors.initials: S
authors.lastname: White
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D006801:Humans; D008024:Ligands; D051381:Rats; D017208:Rats, Wistar; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D008024:Ligands; D018336:Receptors, Aryl Hydrocarbon; D011994:Recombinant Proteins